Estudo piloto de triagem usando a Análise da Curva de Dissociação de Alta Resolução Sensível à Metilação (MS -HRM) em swabs orais para detectar a Síndrome de Prader-Willi / Pilot screening study using Methylation Sensitive High Resolution Dissociation Curve Analysis (MS -HRM) on oral swabs to detect Prader-Willi Syndrome

A síndrome de Prader-Willi (SPW) é uma desordem genética complexa, caracterizada por deleções, dissomia uniparental materna ou defeito no centro de imprinting no alelo paterno do cromossomo 15. As perdas de funções de genes específicos da região 15q11 afetam múltiplos sistemas corporais. O diagnóstico da SPW é difícil de ser realizado com base apenas no exame clínico e envolve a realização de diversas técnicas de biologia molecular para a completa elucidação da etiologia genética, tornando todo o processo laborioso, demorado e custoso. A realização de um teste molecular que permita um diagnóstico rápido e preciso é de vital importância para um melhor prognóstico para paciente. A coleta bem-sucedida de amostras e a extração de DNA de swabs são alternativas não invasivas e confiáveis, tanto para os pacientes quanto para os profissionais que realizarão a coleta destas amostras. Neste trabalho foi possível demonstrar um método simples de coleta de amostras e extração de DNA, que possui baixo custo, é eficaz, fácil e rápido, que fornece uma quantidade e qualidade suficiente de DNA para a execução do MS-HRM, qPCR e sequenciamento. Uma comparação dos procedimentos de extração mostra que o método simples de extração de NaCl é o mais adequado para extração de DNA de amostra bucal coletada através de swab. Neste trabalho foi demonstrado um método simples de coleta de amostras através do swab e extração de DNA com baixo custo e boa qualidade do DNA.

Prader-Willi syndrome (PWS) is a complex disorder, uniparental by deletions, dissociated from no imprint defect in any of the chromosomes 15. As gene variants of the genetic region of the 15q11 region, the diagnosis of PWS is challenging to perform based on clinical examination alone. It involves the performance of several molecular biology techniques for the complete elucidation of genetics, determining the entire laborious, time-consuming, and costly process. The performance of a molecular test allows a quick diagnosis, which is vital for a better prognosis for the patient. Successful sample collection and DNA collection from swabs are non-invasive alternatives for patients and practitioners performing probable sample collection. In this work, it was possible to demonstrate a simple sample collection and DNA method, which has a low cost, is effective, easy, and fast, and provides a sufficient quantity and quality of DNA for the execution of MS-HRM, qPCR, and sequencing. A comparison of the extraction procedures shows that the simple NaCl extraction method is the most appropriate for extracting DNA from a buccal sample collected via swab. In this work, a simple method for swab sampling and extracting DNA was demonstrated, with low cost and good DNA quality.

Evaluación orofacial de los niños Síndrome Prader-Willi / Avaliação orofacial de crianças com Síndrome de Prader-Willi / Orofacial assessment of children with Prader-Willi Syndrome

Resumen Objetivo: Evaluar el estado de salud bucal y el crecimiento craneofacial de pacientes con síndrome de Prader-Willi (SPW), en comparación con niños obesos que no padecen SPW. Métodos y resultados: Se seleccionaron 40 niños con SPW y 40 controles obesos de 10,9 años de edad (control: 11,89 años) y un IMC de 22,72 kg / m2 (control de 36,43 kg / m2). La evaluación de la salud oral ha incluído el número de dientes, tipo de dentición, presencia de caries, sangrado gingival, maloclusión, acumulación de placa, erosión dental, hiperplasia gingival e hipoplasia del esmalte. Los cuestionarios evaluaron los hábitos de higiene oral. Las radiografías panorámicas evaluaron el crecimiento craneofacial. El grupo de casos tenía un 6,8% menos de dientes en comparación con el grupo de control. Se observó una diferencia estadísticamente significativa en el sangrado gingival, la erosión dental y la hipoplasia del esmalte (p = 0,009; p = 0,02 y p = 0,006, respectivamente). Aunque no hubo diferencias estadísticamente significativas, se observó un número aumentado de lesiones cariosas y apiñamiento dental en niños con SPW (p = 0,35 y p = 0,07). Ambos grupos mostraron mala higiene dental. Los niños con SPW mostraron un crecimiento de la rama mandibular aumentada en comparación con el control (p = 0.03). Conclusión: Los niños con SPW tenían hemorragia gingival estática aumentada e hipoplasia del esmalte que los controles con obesidad no SPW. Los niños con SPW pueden presentar un crecimiento vertical craneofacial

Objetivo: Avaliar a saúde bucal e o crescimento craniofacial de pacientes pediátricos com SíndromedePrader-Willi(SPW),emcomparação a crianças obesas não-sindrômicas. Métodos e resultados: Foram selecionadas 40 crianças com SPW e 40 controles não obesos com SPW, com idade de 10,9 anos (controle: 11,89 anos) e IMC 22,72 kg / m2 (controle 36,43 kg / m2). Foram avaliados o número de dentes, tipo de dentição, presença de cárie, sangramento gengival, má oclusão, acúmulo de placa bacteriana, erosão dentária, hiperplasia gengival e hipoplasia do esmalte. Os questionários avaliaram os hábitos de higiene bucal. Radiografias panorâmicas avaliaram o crescimento craniofacial. O grupo caso teve um número 6,8% menor de dentes em comparação ao grupo controle. Observouse diferença estatisticamente significante no sangramento gengival, erosão dentária e hipoplasia do esmalte (p = 0,009; p = 0,02 e p = 0,006, respectivamente). Não houve diferença estatisticamente significante, observou-se um número aumentado de lesões de cárie e apinhamento dentário em crianças com SPW (p = 0,35 e p = 0,07). Ambos os grupos apresentaram má higiene dental. As crianças com SPW apresentaram crescimento aumentado do ramo mandibular com diferença estatisticamente significante (p = 0,03). Conclusão: As crianças com SPW apresentaram sangramento gengival estatisticamente aumentado e hipoplasia do esmalte do que os controles não obesos com SPW. Crianças com SPW podem apresentar crescimento vertical craniofacial aumentado. Mais investigações são necessárias para essa população.

Aim: To assess the oral health status and craniofacial growth of patients with Prader-Willi Syndrome (PWS), compared to obese non-PWS children controls. Methods and Result: 40 PWS children and 40 non-PWS obese controls, aged 10.9 years (control: 11.89 years) and BMI 22.72 kg/m2 (control 36.43 kg/m2) were selected. The number of teeth, type of dentition, presence of caries, gingival bleeding, malocclusion, plaque accumulation, dental erosion, gingival hyperplasia, and enamel hypoplasia were assessed. Questionnaires assessed oral hygiene habits. Panoramic radiographs assessed craniofacial growth. The study group had a 6.8% lower number of teeth compared to the control group. A statistically significant difference was seen in gingival bleeding, dental erosion and enamel hypoplasia (p=0,009; p=0,02 and p=0,006; respectively). There were no statistically significant differences, it was observed an augmented number of carious lesions and Although a higher prevalence of carious lesions and dental crowding was observed in PWS children, the difference was not satisctically significant (p=0.35 and p=0.07 respectively). Both groups showed poor dental hygiene. PWS children showed augmented mandibular ramus growth with a statistically significant difference (p=0.03). Conclusion: PWS children had statically augmented gingival bleeding and enamel hypoplasia than non-PWS obese controls. PWS children may present increased craniofacial vertical growth. Further investigations are needed for this population.

Correlação entre o genótipo e o fenótipo dos pacientes com a Síndrome de Prader-Willi / Correlation between the genotype and phenotype of patients with Prader-Willi Syndrome

A síndrome de Prader-Willi (SPW) é uma doença multissistêmica, cujas manifestações principais incluem hipotonia, obesidade, leve atraso mental, hipogonadismo e insuficiência do hormônio de crescimento. A SPW foi a primeira desordem genética descrita envolvida com o imprinting genômico. O imprinting genômico é uma modificação epigenética do DNA responsável por metilar as ilhas CpG, presentes em regiões promotoras dos genes, inativando a expressão deste gene. Na SPW, o indivíduo possui o alelo materno quimicamente inativado através do imprinting, além disso, o indivíduo perde a função dos mesmos genes no alelo paterno devido a 3 possíveis mecanismos genéticos: Deleção, dissomia uniparental materna (DUM), e microdeleções ou defeitos no centro de controle do imprinting. Ainda há muito a se entender sobre as bases genéticas da SPW e sua correlação com os fenótipos clínicos vistos nestes pacientes, e esta comparação entre o perfil molecular e os sintomas clínicos vistos em pacientes com a SPW vem sendo um tema muito discutido dentro da literatura. Muitos achados suportam uma possível correlação entre o genótipo e o fenótipo destes pacientes. Estabelecendo uma possível correlação entre genótipo e fenótipo irá trazer uma maior compreensão da SPW, provendo um melhor aconselhamento genético e consequentemente melhorando o prognóstico para estes indivíduos e suas famílias. Este estudo tem como objetivo identificar a associação dos diferentes mecanismos genéticos da SPW com os diversos sintomas clínicos, contribuindo para um melhor prognóstico da doença. Um estudo descritivo de pesquisa básica e quantitativa a partir de amostras de sangue periférico de 45 pacientes com padrão de metilação compatível com a SPW acompanhados no Centro de Genética Médica do IFF/FIOCRUZ e pelo Instituto Estadual de Diabetes e Endocrinologia do Estado do rio de Janeiro (IEDE/RJ). O estudo vemsendo desenvolvido no Laboratório de Alta Complexidade do IFF (LACIFF). A abordagem metodológica consistiu no rastreamento destes 45 pacientes utilizando a técnica de MS HRM; MS-MLPA visando identificar as deleções nos pacientes; e o sequenciamento de Sanger visando identificar as dissomias uniparentais maternas e os defeitos no centro de controle do imprinting. Posteriormente foi realizado a coleta de dados fenotípicos dos pacientes que apresentaram alterações compatíveis com a SPW. O trabalho em questão tem como resultados esperados encontrar uma correlação genótipo ­ fenótipo, visando um melhor entendimento sobre as bases genéticas da síndrome e um melhor prognóstico para estes pacientes e suas famílias.

Prader-Willi syndrome (PWS) is a multisystemic disease, the main manifestations of which include hypotonia, obesity, mild mental retardation, hypogonadism, and insufficient growth hormone. SPW was the first described genetic disorder involved with genomic imprinting. Genomic imprinting is an epigenetic modification of the DNA responsible for the methylation of the CpG islands, present in promoter regions of the genes, inactivating the expression of this gene. In PWS, the individual has the maternally allele chemically inactivated through imprinting, in addition, the individual loses the function of the same genes in the paternal allele due to 3 possible genetic mechanisms: Deletion, maternal uniparental disomy (matUPD), and micro deletions or defects in the imprinting control center. There is still a lot to understand about the genetic bases of PWS and its correlation with the clinical phenotypes seen in these patients, and this parallel between the molecular profile and the clinical symptoms seen in patients with PWS has been a very discussed topic in the literature. Many findings support a possible correlation between the genotype and phenotype of these patients. Establishing a possible correlation between genotype and phenotype, will bring a greater understanding of PWS, providing better genetic counseling and consequently improving the prognosis for these individuals and their families. This study aims to identify the association of the different genetic mechanisms of PWS with the different clinical symptoms, contributing to a better prognosis of the disease. A descriptive study of basic and quantitative research based on peripheral blood samples from 45 patients with methylation status compatible with PWS followed at the Medical Genetics Center of IFF / FIOCRUZ and by the State Institute of Diabetes and Endocrinology of the State of Rio de Janeiro (IEDE / RJ). The study has been developed at the IFF High Complexity Laboratory (LACIFF). The methodological approach consisted of tracking these 45 patients using the MS-HRM technique; MS MLPA to identify deletions in patients; and the Sanger sequencing aiming to identify maternal uniparental dissomies and defects in the imprinting control center. Subsequently, phenotypic data were collected from patients who presented changes compatible with PWS. The work in question has as expected results to find this genotype - phenotype correlation, aiming at a better understanding about the genetic bases of the syndrome and a better prognosis for these patients and their families.

Prader-Willi syndrome: endocrine manifestations and management

ABSTRACT Prader-Willi syndrome (PWS) is a genetic disorder caused by the absence of gene expression in the 15q11.2-q13 paternal chromosome. Patients with PWS develop hypothalamic dysfunction that can lead to various endocrine changes such as: obesity, growth hormone deficiency, hypogonadism, hypothyroidism, adrenal insufficiency and low bone mineral density. In addition, individuals with PWS have increased risk of developing type 2 diabetes mellitus. This review summarizes and updates the current knowledge about the prevention, diagnosis and treatment of endocrine manifestations associated with Prader Willi syndrome, especially diagnosis of growth hormone deficiency, management and monitoring of adverse effects; diagnosis of central adrenal insufficiency and management in stressful situations; screening for central hypothyroidism; research and treatment of hypogonadism; prevention and treatment of disorders of glucose metabolism. Careful attention to the endocrine aspects of PWS contributes significantly to the health of these individuals. Arch Endocrinol Metab. 2020;64(3):223-34

Clinical screening and genetic diagnosis for Prader-Willi syndrome / 中国当代儿科杂志

OBJECTIVE@#To study the clinical screening and genetic diagnosis of children suspected of Prader-Willi syndrome (PWS), as well as the differences in the scores of clinical diagnostic criteria among the children with a confirmed diagnosis of PWS.@*METHODS@#A total of 94 children suspected of PWS who were admitted from July 2016 to December 2018 were enrolled as subjects. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to confirm the diagnosis. For the children with a confirmed diagnosis of PWS, the scores of clinical diagnostic criteria were determined, and the perinatal characteristics were analyzed.@*RESULTS@#A total of 11 children with PWS were confirmed by MS-MLPA, with a detection rate of 12%, among whom there were 7 boys and 4 girls, with a median age of 3 years and 4 months (range 25 days to 6 years and 8 months) at the time of confirmed diagnosis. Among the 11 children with PWS, only 5 children (45%) met the criteria for clinical diagnosis. The main perinatal characteristics of the children with PWS were decreased fetal movement (9 cases, 82%), cesarean section birth (11 cases, 100%), hypotonia (11 cases, 100%), feeding difficulties (11 cases, 100%), and weak crying (11 cases, 100%).@*CONCLUSIONS@#Gene testing should be performed as early as possible for children suspected of PWS by clinical screening. PWS may be missed if only based on the scores of clinical diagnostic criteria.

Edição genômica como estudo da regulação da expressão do hormônio de crescimento na síndrome de Prader-Willi / Genomic editing as a study of the regulation of growth hormone expression in Prader-Willi syndrome

A síndrome de Prader-Willi (SPW) é associada a distúrbios neurológicos, comportamentais e diversas deficiências hormonais, incluindo o hormônio do crescimento (GH). Embora o tratamento de reposição do GH melhore a composição corporal, o crescimento e o quadro clínico geral, esta não é uma cura e suas bases clínicas ainda são desconhecidas na síndrome. A SPW ocorre por três mecanismos moleculares: deleção paterna da região 15q11-q13; dissomia uniparental materna 15; ou defeitos de imprinting. A linhagem celular GH3 de pituitária de rato foi utilizada por ser um modelo bem estudado de células secretoras de GH e prolactina. Utilizando o sistema CRISPR-Cas9, realizamos a deleção completa da região ortóloga da SPW de 3,2 Mb na linhagem GH3, produzindo o genótipo da síndrome in vitro, para investigar quais segmentos gênicos da SPW estão envolvidos na regulação do GH. A investigação de off-targets por sequenciamento Sanger revelou desde reparos sem alteração nucleotídica até grandes rearranjos nos flancos dos sítios-alvo dos gRNAs, inclusive com inserções não previstas de DNA exógeno. Nossos dados ressaltam a necessidade de projetar cuidadosamente as condições experimentais e caracterizar minuciosamente os materiais genéticos obtidos pelo sistema CRISPR-Cas9. A análise do DNA no flanco proximal da região da SPW em ratos demonstrou sequências que podem representar o marco inicial do silenciamento gênico por imprinting na região, assim como ocorre próximo a UBE3A em humanos na porção distal da região ortóloga. Os quatro modelos de sublinhagens SPW-Knockout gerados neste trabalho com deleções de 3,2 Mb envolvendo toda a região da SPW permitirão compreender melhor a relação entre os genes da síndrome e as vias moleculares envolvidas na regulação do GH, além da sua utilização como modelos em novos estudos sobre a SPW. O gene SNORD107 surgiu como o principal candidato a regular o GH dentro da região da SPW, podendo formar um complexo ribonucleoprotéico que exerceria função regulatória pós-transcricional na cadeia de produção do GH.

Prader-Willi syndrome (PWS) is associated with neurodevelopmental and behavioral abnormalities and numerous hormonal deficiencies, including growth hormone (GH). Although GH replacement treatment improves body composition, growth, and the overall clinical presentation, it is not a cure and its clinical basis is still unknown in the syndrome. PWS occurs by three molecular mechanisms: paternal deletion of the 15q11-q13 region; maternal uniparental disomy 15; or imprinting defects. The rat pituitary GH3 cell line is a well-studied model of GH and prolactin-secreting cells. Using the CRISPR-Cas9 system, we performed the complete deletion of the 3.2 Mb PWS orthologous region in GH3 cells, generating the syndrome genotype in vitro to investigate which PWS genes are involved in GH regulation. Off-target analysis by Sanger sequencing revealed perfect breakpoint repairs, but also large rearrangements on the flanking sites of the gRNA targets, including unexpected insertions of exogenous DNA. These data highlight the need to carefully design the experimental conditions and fully characterize the genetic materials obtained by CRISPR-Cas9. DNA analysis on the proximal flank of the PWS region in rats has shown sequences that can mark the initial borders of genomic imprinting, as it occurs close to UBE3A in humans in the distal portion of the orthologous region. The four PWS-Knockout models generated in this work with 3.2 Mb deletions involving the entire PWS region will allow a better understanding of the relationship between PWS genes and the molecular pathways involved in GH regulation and can be used as models in new PWS studies. The SNORD107 gene has emerged as the main candidate to regulate GH within the PWS region and may form a ribonucleoprotein complex with a post-transcriptional regulatory function in the GH production pathway.

Intervención multidisciplinaria en paciente diagnosticado con el síndrome de Prader-Will / Interdisciplinary intervention in a pacient diagnosed with Prader-Willi síndrome

Introducción: El síndrome de Prader-Willi es una enfermedad genética, causada por deleciones de novo en la región 15q11q13 en el cromosoma paterno. Se caracteriza por falta de saciedad que conduce a obesidad mórbida, trastornos del comportamiento, discapacidad intelectual, baja estatura e hipogonadismo. Objetivo: Describir los resultados obtenidos del análisis e intervención multidisciplinar realizados en paciente diagnosticado con el síndrome de Prader-Willi. Presentación del caso: Análisis de caso clínico en menor de 8 años, sexo masculino, diagnosticado con síndrome de Prader-Willi, a través de intervención multidisciplinaria realizado en tres momentos: evaluación, diagnóstico e intervención con enfoque cognitivo conductual. Conclusiones: Las estrategias adoptadas generaron cambios significativos en el contexto familiar y social, entre ellas, apropiación de las recomendaciones suministradas, adopción de factores protectores, identificación de roles y optimización en la adherencia farmacológica. La atención a estas consideraciones proporciona mejoras, que apuntan a la calidad de vida y clínica del paciente(AU)

Introduction: Prader-Willi syndrome is a genetic disease caused by de novo deletions in the 15q11q13 region in the paternal chromosome. It is characterized by lack of satiety leading to morbid obesity, behavioral disorders, intellectual disability, short height and hypogonadism. Objective: To describe the results obtained from the multidisciplinary analysis and intervention performed in a patient diagnosed with Prader-Willi syndrome. Presentation of the clinical case: Clinical case analysis in an 8 years old child, male sex, diagnosed with Prader-Willi syndrome through a multidisciplinary intervention performed in three moments: assessment, diagnosis and intervention with cognitive behavioral approach. Conclusions: The strategies adopted generated significant changes in the social and family context, family´s appropriation of the recommendations provided, adoption of protective factors, roles identification and improving of adherence to treatment. By taking into account this considerations, improvements lead to clinic and life quality of the patient(AU)

Chromosomal Microarray Analysis as a First-Tier Clinical Diagnostic Test in Patients With Developmental Delay/Intellectual Disability, Autism Spectrum Disorders, and Multiple Congenital Anomalies: A Prospective Multicenter Study in Korea

BACKGROUND: To validate the clinical application of chromosomal microarray analysis (CMA) as a first-tier clinical diagnostic test and to determine the impact of CMA results on patient clinical management, we conducted a multicenter prospective study in Korean patients diagnosed as having developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA). METHODS: We performed both CMA and G-banding cytogenetics as the first-tier tests in 617 patients. To determine whether the CMA results directly influenced treatment recommendations, the referring clinicians were asked to complete a 39-item questionnaire for each patient separately after receiving the CMA results. RESULTS: A total of 122 patients (19.8%) had abnormal CMA results, with either pathogenic variants (N=65) or variants of possible significance (VPS, N=57). Thirty-five well-known diseases were detected: 16p11.2 microdeletion syndrome was the most common, followed by Prader-Willi syndrome, 15q11-q13 duplication, Down syndrome, and Duchenne muscular dystrophy. Variants of unknown significance (VUS) were discovered in 51 patients (8.3%). VUS of genes putatively associated with developmental disorders were found in five patients: IMMP2L deletion, PTCH1 duplication, and ATRNL1 deletion. CMA results influenced clinical management, such as imaging studies, specialist referral, and laboratory testing in 71.4% of patients overall, and in 86.0%, 83.3%, 75.0%, and 67.3% of patients with VPS, pathogenic variants, VUS, and benign variants, respectively. CONCLUSIONS: Clinical application of CMA as a first-tier test improves diagnostic yields and the quality of clinical management in patients with DD/ID, ASD, and MCA.

Genetic diagnosis and non-invasive prenatal testing of a fetus with Prader-Willi/Angelman syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis for a fetus featuring growth restriction and validate the effectiveness of a novel noninvasive prenatal testing (NIPT) technique for the detection of chromosomal microdeletions.@*METHODS@#Next-generation sequencing(NGS) and fluorescence in situ hybridization(FISH) were used to analyze the DNA of the fetus. Conventional G-banding was used to analyze the karyotypes of the fetus and its parents. High-throughput sequencing was used to analyze free fetal DNA.@*RESULTS@#NGS analysis has revealed a 4.88 Mb deletion at 15q11.2-q13.1 region in the fetus, which has a 99% overlap with the critical region of Prader-Willi syndrome (Type 2) and Angelman syndrome (Type 2) and encompassed critical genes including SNRPN and UBE3A. NIPT also revealed a 4.6 Mb deletion at 15q12, which was consistent with the results of fetal cord blood and amniotic DNA testing. FISH assay has confirmed the result of NGS. By karyotying, all subjects showed a normal karyotypes at a level of 320~400 bands.@*CONCLUSION@#It is quite necessary to carry out genetic testing on fetuses showing growth restriction. NIPT for fetal chromosomal microdeletions/microduplication syndromes is highly accurate for the diagnosis of Prader-Willi/Angelman syndrome.

Aplicação do sequenciamento de nova geração no diagnóstico molecular da síndrome de Prader-Willi / Application of next-generation sequencing in the molecular diagnosis of Prader-Willi syndrome

Introdução: A síndrome de Prader- Willi (SPW) é uma desordem genética complexa, caracterizada por deleções, dissomia uniparental materna ou defeito no centro de imprinting no alelo paterno do cromossomo 15. As perdas de funções de genes específicos da região 15q11 afetam múltiplos sistemas corporais. O diagnóstico da SPW envolve a realização de diversas técnicas de biologia molecular e citogenética para a completa elucidação do mecanismo genético relacionado ao desenvolvimento da síndrome, tornando todo o processo laborioso, demorado e custoso. Objetivo: Aplicação da tecnologia sequenciamento de nova geração (NGS), plataforma Ion Torrent PGM, para diagnostico molecular da SPW. Materiais e Métodos: Foram incluídos 17 pacientes suspeitos para SPW onde foram submetidos a análise de metilação (MS-HRM), citogenética (GTG e FISH) e sequenciamento de genes alvos na plataforma Ion Torrent PGM, e subsequente confirmação de variantes através da técnica de sequenciamento de Sanger. Resultados: A análise de metilação detectou 6 indivíduos portadores da SPW, 1 portador da Síndrome de Angelman (SA) e 10 indivíduos normais. A técnica de GTG identificou 1 indivíduo com uma grande perda cromossômica, já a metodologia de FISH identificou 3 indivíduos com deleções, totalizando 4. O bom desempenho da tecnologia de sequenciamento NGS, através da metodologia Ion Torrent PGM, permitiu a realização de análises de frequência alélica, variação do número de cópias (CNV) e de mutações específicas do genoma. As análises por bioinformática realizadas nos dados do NGS permitiram detectar 4 pacientes portadores de deleção, classificando-as como Tipo 1 ou Tipo 2. Além disso, foi possível identificar um evento raro de dissomia uniparental segmentar, com complicações prognósticas severas. Identificou-se variantes do tipo INDEL no gene PWRN1 em 2 pacientes, onde, o impacto funcional desta mutação ainda não foi estudado. Contudo o gene possui forte correlação com o desenvolvimento da SPW. A metodologia também identificou uma mutação INDEL no gene MAGEL2 em um paciente com padrão de metilação normal no MS-HRM, sugerindo a identificação de uma síndrome análoga a SPW. Todas as variantes detectadas foram validadas através do sequenciamento de Sanger. Conclusão: A plataforma Ion Torrent identificadou todas as alterações relacionadas ao desenvolvimento da SPW. O pipeline desenvolvido mostrou-se aplicável a uma rotina diagnóstica.

Introduction: Prader-Willi syndrome (PWS) is a complex genetic disorder characterized by deletions, maternal uniparental disomy or defect at the imprinting center in the paternal allele of chromosome 15. Loss of 15q11 region-specific gene functions affects multiple body systems. The diagnosis of SPW involves the accomplishment of several techniques of molecular biology and cytogenetics for the complete elucidation of the genetic mechanism related to the development of the syndrome, making the whole process laborious, time-consuming and costly. Objective: Application of the Next-Generation sequencing technology (NGS), platform Ion Torrent PGM, for molecular diagnosis of PWS. Materials and Methods: Seventeen suspected patients for SPW were submitted to methylation (MS-HRM), cytogenetic analysis (GTG and FISH) and sequencing of target genes in the Ion Torrent PGM platform, and subsequent confirmation of variants by the technique of sequencing of Sanger. Results: Methylation analysis detected 6 individuals with SPW, 1 with Angelman Syndrome (AS) and 10 normal individuals. The GTG technique identified 1 individual with a large chromosomal loss, and the FISH methodology identified 3 individuals with deletions, totaling 4. The good performance of the NGS sequencing technology, through the Ion Torrent PGM methodology, allowed the performance of frequency analyzes allelic, copy number variation (CNV), and genome-specific mutations. The bioinformatics analyses performed on the NGS data allowed the detection of 4 patients with deletion, classifying them as Type 1 or Type 2. In addition, it was possible to identify a rare segmental uniparental disomy with severe prognostic complications. Variables of the INDEL type were identified in the PWRN1 gene in 2 patients, where the functional impact of this mutation has not been studied. However, the gene has a strong correlation with the development of PWS. The methodology also identified an INDEL mutation in the MAGEL2 gene in a patient with normal methylation pattern in MS-HRM, suggesting the identification of a syndrome similar to PWS. All detected variants were validated through Sanger sequencing. Conclusion: The Ion Torrent platform has identified all the changes related to the development of PWS. The pipeline developed proved to be applicable to a diagnostic routine.

Anestesia locorregional en una paciente adulta con síndrome de Prader-Willi: consideraciones anestésicas / Anesthetic consideration: Regional anesthesia with Prader-Willi

Prader-Willi syndrome (PWS) is a disorder caused by a genetic alteration that causes a multisystem clinic. It can be due mainly to three genetic mechanisms; a paternal deletion of the 15q11-13 region, a maternal uniparental disomy, or an imprinting defect. The paternal deletion is observed in 70% of the patients, the disomy in 25% and the imprinting defect in only 5% of those affected by this syndrome. 1) It is the most common syndromic cause of obesity with an estimated prevalence in the population of 1: 50,000; 2) The clinic is very variable, which is why clinical criteria have been created that, supported by the genetic study, confirm the diagnosis; 3) They have difficulty feeding during lactation, which leads to hyperphagia in childhood that leads to obesity. In the adult stage, in addition to obesity, respiratory pathology, sleep disturbances and psychological disorders stand out; 4) Objective: the aim of the present review was to compile the cases recorded in the scientific literature of patients anesthetized with PWS and the anesthetic options used in said patients.

El síndrome de Prader-Willi (SPW) es un trastorno causado por una alteración genética que provoca una clínica multisistémica. Puede ser debido principalmente a tres mecanismos genéticos; una deleción paterna de la región 15q11-13, una disomía uniparental materna o un defecto de impronta. La deleción paterna se observa en el 70% de los pacientes, la disomía en el 25% y el defecto de impronta en tan solo el 5% de los afectados por este síndrome. 1) Constituye la causa sindrómica más frecuente de obesidad con una prevalencia estimada en la población de 1:50.000; 2) La clínica es muy variable por lo que se han creado unos criterios clínicos que apoyados por el estudio genético confirman el diagnóstico; 3) Presentan dificultad para la alimentación durante la lactancia, que da paso a una hiperfagia en la infancia que deriva en obesidad. En la etapa adulta, además de la obesidad destacan la patología respiratoria, alteraciones del sueño y trastornos psicológicos; 4) Objetivo: el objetivo de la presente revisión fue recopilar los casos registrados en la literatura científica de pacientes anestesiados con SPW y las opciones anestésicas utilizadas en dichos pacientes.

Síndrome de prader willi: o que o pediatra geral deve fazer - uma revisão / Prader-willi syndrome: what is the general pediatrician supposed to do? - a review

RESUMO Objetivo: Realizar uma revisão sobre a Síndrome de Prader-Willi (SPW) com base nas publicações mais recentes e fornecer recomendações ao pediatra geral para diagnóstico precoce e seguimento. Fonte de dados: Artigos publicados nas bases Pubmed e SciELO. A pesquisa não foi limitada a um período e incluiu todos os artigos das bases de dados. Síntese dos dados: A SPW é uma síndrome genética rara, resultante da perda do imprinting gênico expresso no cromossomo paterno 15q11-q13, sendo caracterizada por alterações endocrinológicas, como deficiência de hormônio de crescimento, obesidade, insuficiência adrenal central, hipotireoidismo, hipogonadismo, além de alterações comportamentais e déficit intelectual. Há outras comorbidades associadas, como distúrbios de sono, escoliose, constipação, problemas dentários e alterações de coagulação. O protocolo de seguimento da SPW do Instituto da Criança da Universidade de São Paulo se baseia em quarto pilares principais: dieta, exercício físico, terapia com hormônio de crescimento humano recombinante (rhGH) e manejo comportamental e cognitivo. A dieta deve ser restrita a 900 kcal/dia, de acordo com a Pirâmide Alimentar do Prader-Willi, e o exercício físico deve ser diário, aeróbico e postural. A terapia com rhGH é fortemente recomendada pela literatura científica internacional e deve ser iniciada assim que for realizado o diagnóstico da síndrome. O manejo do comportamento é realizado com estratégias para estabelecer rotina e regras. Conclusões: Se a SPW se tornar mais familiar ao pediatra geral, o diagnóstico e o tratamento começarão mais precocemente, o que irá melhorar a qualidade de vida e os cuidados desses pacientes.

ABSTRACT Objective: To carry out a review about Prader-Willi Syndrome based on the most recent data about the subject and to give recommendation for the general pediatricians for early diagnoses and follow-up. Data sources: Scientific articles in the PubMed and SciELO databases. The research was not limited to a specific time period and included all articles in such databases. Data synthesis: The Prader-Willi Syndrome (PWS) is a rare genetic disorder resulting from the loss of imprinted gene expression within the paternal chromosome 15q11-q13. PWS is characterized by endocrine abnormalities, such as growth hormone (GH) deficiency, obesity, central adrenal insufficiency, hypothyroidism, hypogonadism and complex behavioral and intellectual difficulties. PWS individuals also may present other comorbidities, such as sleep disorders, scoliosis, constipation, dental issues and coagulation disorders. The follow-up protocol of the Children's Institute at Universidade de São Paulo is based on four main pillars: diet, exercise, recombinant human growth hormone (rhGH) therapy and behavioral and cognitive issues. The diet must include a caloric restriction of 900 kcal/day, according to the Prader-Willi Eating Pyramid and exercise plan is focused on daily aerobic exercises and postural therapy. The rhGH therapy is highly recommended by the international scientific literature and must be started as soon as the diagnostic is made. The management of behavioral issues is based on strategies to establish routine and rules. Conclusions: If the general pediatrician becomes more familiar with PWS, the diagnosis and treatment will start earlier, which is essential to improve the quality of life and care for these individuals.

Bariatric surgery in individuals with severe cognitive impairment: report of two cases / Cirurgia bariátrica em indivíduos com déficits cognitivos graves: relato de dois casos

ABSTRACT CONTEXT: Bariatric surgery has become the gold-standard treatment for refractory morbid obesity. Obesity is frequently associated with certain syndromes that include coexisting cognitive deficits. However, the outcomes from bariatric surgery in this group of individuals remain incompletely determined. CASE REPORT: A 25-year-old male with Prader-Willi syndrome, whose intelligence quotient (IQ) was 54, was admitted with a body mass index (BMI) of 55 kg/m2, associated with glucose intolerance. He underwent the Scopinaro procedure for biliopancreatic diversion, with uneventful postoperative evolution, and presented a 55% loss of excess weight one year after the surgery, with resolution of glucose intolerance, and without any manifestation of protein-calorie malnutrition. A 28-year-old male with Down syndrome, whose IQ was 68, was admitted with BMI of 41.5 kg/m2, associated with hypertension. He underwent Roux-en-Y gastric bypass, with uneventful postoperative evolution. He presented a 90% loss of excess weight one year after the surgery, with resolution of the hypertension. CONCLUSION: Bariatric surgery among individuals with intellectual impairment is a controversial topic. There is a tendency among these individuals to present significant weight loss and comorbidity control, but less than what is observed in the general obese population. The severity of the intellectual impairment may be taken into consideration in the decision-making process regarding the most appropriate surgical technique. Bariatric surgery is feasible and safe among these individuals, but further research is necessary to deepen these observations.

RESUMO CONTEXTO: A cirurgia bariátrica tornou-se o tratamento padrão ouro para a obesidade mórbida refratária. A obesidade está frequentemente associada a certas síndromes nas quais também coexistem déficits cognitivos, entretanto, os resultados da cirurgia bariátrica nesse grupo de indivíduos ainda não foram completamente determinados. RELATO DE CASO: Um homem de 25 anos com síndrome de Prader-Willi, cujo quociente de inteligência (QI) era estimado em 54, foi admitido com índice de massa corporal (IMC) de 55 kg/m2, associado com intolerância à glicose. Foi submetido a uma derivação biliopancreática à Scopinaro, com evolução pós-operatória sem complicações significativas. Apresentou perda de 55% do excesso de peso um ano após a cirurgia, com resolução da intolerância à glicose, sem manifestação de desnutrição proteico-calórica. Outro paciente, homem de 28 anos com syndrome de Down, cujo QI era de 68, foi admitido com IMC de 41,5 kg/m2, associado a hipertensão arterial. Foi submetido ao bypass gástrico em Y de Roux, com evolução pós-operatória sem complicações. Apresentou perda de 90% do excesso de peso após um ano e resolução da hipertensão. CONCLUSÃO: A cirurgia bariátrica em indivíduos com déficits intelectuais é um tópico controverso. Existe uma tendência entre esses indivíduos de apresentar perda de peso e controle de comorbidades significativos, porém menores que os observados na população obesa geral. A gravidade do déficit intelectual pode ser considerada no processo de decisão sobre a técnica cirúrgica mais adequada. A cirurgia bariátrica é factível e segura nesse grupo de indivíduos. Porém, mais estudos são necessários para aprofundar estas observações.

Análisis de variación del número de copias y de patrones de metilación en la región 15q11-q13 / Variation analysis of the number of copies and methylene patterns in region 15q11-q13

La región q11-q13 del cromosoma 15 humano es proclive a sufrir alteraciones genéticas. Algunos genes de la región presentan expresión parental diferencial monoalélica, regulada por imprinting (EI). Errores en la regulación del EI, disomías uniparentales (DSU), así como también el cambio en el número de copias genómicas (CNV) producidos por sitios susceptibles de quiebre cromosómico (BP), producen alteraciones en esta región. Las enfermedades más frecuentes asociadas son el síndrome de Prader-Willi, el síndrome de Angelman y el síndrome de microduplicación 15q11-q13. En el presente trabajo analizamos la región 15q11-q13 por Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA) en 181 muestras de ADN derivadas a nuestro servicio de análisis genético molecular. En este trabajo mostramos que, de las 181 muestras, 39 presentaron alteraciones detectables por MS-MLPA. El 61.5% (24/39) de esas alteraciones detectadas fueron deleciones, el 5.1% (2/39) duplicaciones y el 33.3%(13/39) DSU/EI. Los CNV fueron 4 veces más frecuentes que las DSU/EI (OR = 4; IC 95%: 1.56-10.25) consistente con la literatura. Entre los CNV, dos casos atípicos permiten postular posibles sitios BP que no han sido informados en la literatura previamente.

Human chromosome 15q11-q13 region is prone to suffer genetic alterations. Some genes of this region have a differential monoallelic imprinting-regulated expression pattern. Defects in imprinting regulation (IE), uniparental disomy (UPD) or copy number variation (CNV) due to chromosomal breakpoints (BP) in 15q11-q13 region, are associated with several diseases. The most frequent are Prader-Willi syndrome, Angelman syndrome and 15q11-q13 microduplication syndrome. In this work, we analyzed DNA samples from 181 patients with phenotypes which were compatible with the above-mentioned diseases, using Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA). We show that, of the 181 samples, 39 presented alterations detectable by MS-MLPA. Of those alterations, 61.5% (24/39) were deletions, 5.1% (2/39) duplications and 33.3% (13/39) UPD/IE. The CNV cases were 4 times more frequent than UPD/IE (OR= 4; IC 95%: 1.56-10.25), consistent with the literature. Among the CNVs, two atypical cases allow to postulate new possible BP sites that have not been reported previously in the literature.

Etiological Classification and Developmental Outcomes in Floppy Infants: A Single Tertiary Center Experience / 대한소아신경학회지

PURPOSE: Floppy infants or congenital hypotonia indicates decreased muscle tone in infants secondary to abnormalities of the central or the peripheral nervous system, or both. Previous literature classified its causes as those attributable to a central vs. peripheral origin; however, recent studies have introduced a newer classification describing a combined origin. We invenstigated floppy infants by applying the new etiological classification and reviewed the most common etiologies based on the age of presentation. We additionally reviewed the clinical characteristics, diagnoses, and the developmental outcomes in these infants. METHODS: We retrospectively reviewed the electronic medical charts and recruited 116 infants diagnosed with floppy infant syndrome between January 2005 and December 2016 at Severance Children's Hospital. Among these infants, 66 with a confirmed diagnosis were reviewed for the etiological classification. Information regarding developmental outcomes was obtained via phone interviews with the infants' families. RESULTS: Based on the new etiological classification, among 69 infants with a confirmed diagnosis, in 40 (34.5%) this syndrome was of central origin, in 19 (16.4%) of peripheral origin, and in 10 (8.6%) of combined origin. Prader-Willi syndrome, myotonic dystrophy, and spinal muscular atrophy were the most common disorders observed and combined hypotonia showed the poorest developmental outcome. CONCLUSION: The study states the importance of proper evaluation of etiological diagnosis and optimal intervention for developmental prognosis. The introduction of a new etiological group of combined hypotonia especially emphasizes regular monitoring and timely rehabilitative intervention in patients for the better quality of life in them as well as their caregivers.

Clinical Usefulness of Aripiprazole Treatment in a Girl with Prader-Willi Syndrome and Psychosis

Prader-Willi syndrome (PWS) is a quite rare multi-systemic genetic disorder strongly associated with psychiatric illness in adults, especially psychosis. This report presents a 16-year-old female with PWS and symptoms of brief psychotic disorder with a complete resolution of symptoms under aripiprazole medication. However, an exacerbation occurred after aripiprazole reduction. Apart from a weight gain of about 2 kg over the course of two years, no adverse effects could be found. This first report on the use of aripiprazole in a subject with PWS and psychosis suggests that aripiprazole might be a promising treatment approach in this distinct group of patients.

Experience with reversal of a neuromuscular block with sugammadex in a child with Prader-Willi syndrome: A case report

We treated a 4-year-old patient with a genetic disorder, Prader-Willi syndrome, that was accompanied by pulmonary hypertension due to upper airway obstruction. Prader-Willi syndrome is a complex genetic condition characterized by hypotonia, feeding difficulties, poor growth, and delayed development. Hypotonia was the main concern in the anesthetic management of this patient, including the choice of a neuromuscular blocking agent. We report successful induction of anesthesia in this patient with sevoflurane inhalation, remifentanil infusion, and a non-depolarizing muscle relaxant, rocuronium, while following up the status of the neuromuscular block by train-of-four monitoring and reversing the neuromuscular block with sugammadex.

Experience with reversal of a neuromuscular block with sugammadex in a child with Prader-Willi syndrome: A case report

We treated a 4-year-old patient with a genetic disorder, Prader-Willi syndrome, that was accompanied by pulmonary hypertension due to upper airway obstruction. Prader-Willi syndrome is a complex genetic condition characterized by hypotonia, feeding difficulties, poor growth, and delayed development. Hypotonia was the main concern in the anesthetic management of this patient, including the choice of a neuromuscular blocking agent. We report successful induction of anesthesia in this patient with sevoflurane inhalation, remifentanil infusion, and a non-depolarizing muscle relaxant, rocuronium, while following up the status of the neuromuscular block by train-of-four monitoring and reversing the neuromuscular block with sugammadex.

Endocrine and metabolic features of female children with Prader-Willi syndrome: an analysis of 4 cases / 中国当代儿科杂志

This article reports the clinical features and endocrine and metabolic features of 4 children with Prader-Willi syndrome (PWS). All the patients were female and aged 6-12 years at diagnosis. All of them had clinical manifestations of obesity, unusual facies, developmental retardation, and intellectual disability. Genetic detection showed that 2 patients had paternal deletion of the 15q11.2-q13 region, one patient had maternal autodiploid in the 15q11.2-q13 region, and one patient had no abnormality in the 15q11.2-q13 region. All patients had varying degrees of endocrine and metabolic disorders: 2 patients had short stature, among whom one had delayed appearance of secondary sex characteristics and the other one had type 2 diabetes; one patient had insulin resistance and no mammary gland development; one patient had a body height of P-Pand precocious puberty. Patients with PWS have various endocrine disorders, so long-term endocrine follow-up and management is very important.

Prenatal diagnosis of a fetus with 5p15.33 microdeletion / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To screen for genomic copy number variants (CNVs) in a fetus with one sibling affected with Prader-Willi syndrome using single nucleotide polymorphism (SNP) array.</p><p><b>METHODS</b>The fetus and its parents were subjected to chromosomal karyotyping and SNP array analysis.</p><p><b>RESULTS</b>A 5p15.33 microdeletions was identified in the fetus and its phenotypically normal mother with a size of 344 kb (113 576 to 457 213). The father was normal for both testing. Analysis of literature and CNVs database indicated the above CNV to be variant of unclear significance. The couple decided to continue with the pregnancy and gave birth to a healthy boy at full-term. No abnormalities were found during the follow-up.</p><p><b>CONCLUSION</b>This study may provide further data for the phenotype-genotype correlation of 5p15.33 microdeletion, which differs from Cri du Chat syndrome.</p>